The development of a clean, sustainable and economically viable energy supply for the future is one of the most urgent challenges of our generation. Oil production is expected to peak in around 5 to 30 years time and economically viable oil reserves will be largely depleted by 2050. More recent reports, however, suggest that oil production may already have peaked in 2000. A viable hydrogen economy requires clean, sustainable and economic ways of generating hydrogen. Current hydrogen production depends almost entirely on the use of non-renewable resources (i.e. steam reformation of natural gas, coal gasification and nuclear power driven electrolysis of water). Although these approaches are initially likely to drive a transition towards a hydrogen economy, the hydrogen produced is more expensive and contains less energy than the non-renewable energy source from which it is derived. In addition, the use of fossil fuels and nuclear power is unsustainable. Therefore, there is a clear need to establish economically viable means of hydrogen production.
A particularly desirable option is the production of hydrogen using photosynthetic organisms, since the ultimate energy source is solar energy. Algal pond technology is likely to be cheap compared to alternatives such as photovoltaic cells. Furthermore and in contrast to all other sustainable energy systems (except biomass), which incur an initial CO2 emissions penalty during manufacture, algae have the advantage that they are able to sequester CO2 while self-assembling their intricate solar collectors. This gives them an additional intrinsic value in terms of carbon trading upon setup.
Solar energy is captured and stored in the form of starch and other molecules including proteins, which are subsequently used as a fuel to drive ATP production via the processes of oxidative phosphorylation in the mitochondria (FIG. 1). Some green algae have evolved the ability to channel the H+ and e− stored in starch into H2 production under anaerobic conditions. Thus there is the promise that hydrogen may be generated using algal bioreactors. In the first step of photosynthesis, Photosystem II (PSII) drives the most highly oxidizing reaction known to occur in biology, splitting H2O into oxygen (O2), protons (H+) and electrons (e−) (FIG. 1). O2 is released into the atmosphere and is responsible for maintaining aerobic life on Earth. The derived e− are passed along the photosynthetic e− transport chain (FIG. 2) from PSII via Plastoquinone (PQ) to Cytochrome b6f (cyt b6f) and Photosystem I (PSI), and are ultimately used in the production of NADPH. In a parallel process (photophosphorylation), H+ are released into the thylakoid lumen (FIG. 1) where they generate a H+ gradient that is used to drive ATP production via ATP synthase. NADPH and ATP are subsequently used to produce starch and other biomolecules.
ATP and NADPH/NADH are fundamental requirements of all living cells. The inhibition of PSII (eg. by incubation in the dark) blocks the supply of H+ and e− that are used to generate ATP and NADPH via photophosphorylation in the chloroplast. For a time, the shortage of ATP and NADPH caused by the inhibition of PSII can be compensated for via aerobic respiration mediated by the mitochondrial e− transport chain (FIG. 1), which metabolises starch, proteins and lipids. As its name suggests, oxidative phosphorylation requires O2. The O2 is combined with H+ and e− by mitochondrial Complex IV to generate H2O, which essentially acts as an H+ and e− sink (FIG. 1). Under anaerobic conditions Complex IV is inhibited, blocking e− transport through the remainder of the e− transport chain consisting of Complexes I, II, III and Cytochrome oxidase (also known as Complex IV). Under strictly anaerobic conditions most photosynthetic organisms die. However, a select number of photosynthetic organisms such as the green alga C. reinhardtii have a third mechanism, which allows them to switch into a mode of ATP and NADPH production (FIG. 2). Under illuminated anaerobic conditions, they generate ATP in the chloroplast while simultaneously producing H2 as a volatile H+/e− sink, instead of H2O. This process involves the Hydrogenase HydA located in the chloroplast stroma (Florin et al., 2001 and Happe and Kaminski, 2002.). HydA transcription and activity is strongly inhibited by O2 (Ghirardi et al., 1997). It is likely that oxygen sensitivity acts as a molecular control switch which tells HydA when anaerobic conditions occur.
In order to optimise ATP and NADPH production under transient light conditions, plants and algae developed a redox-controlled regulation mechanism called the LHC state transitions. This process normally balances PSI and PSII turnover rates by regulating the size of their light harvesting antennae (LHCI & LHCII, respectively), specifically by shuttling Lhcb proteins between the two photosystems (State 1: large PSII antenna; State 2: large PSI antenna). In the green alga C. reinhardtii, this process results in a switch from linear to cyclic photosynthetic electron transport, which could compete with the Fe-hydrogenase HydA for e− at the reducing side of PSI. Cells blocked in state 1 under anaerobic conditions do not perform cyclic electron transfer in which electrons are transferred back to Cytb6f. Under these conditions the Fe-hydrogenase HydA no longer has to compete with Cytb6f mediated cyclic electron transport for the electrons derived from PSI.
The earliest reports of algal H2 production date back to the 1930s (Stephenson and Stickland, 1931). It was discovered that certain green algae and cyanobacteria could produce H2 gas upon illumination, by a reaction that was extremely sensitive to inhibition by O2. Despite the obvious attraction of using photosynthetic organisms for sustainable H2 production from H2O, it was not until 2000 that Melis and co-workers first reported a method to overcome this inhibition (Melis, 2000 and U.S. Patent Application No. 2001/005343). Melis describes a process in which the inhibition was lifted by temporally separating the O2 generating H2O splitting reaction, catalysed by PSII, from the O2 sensitive H2 production catalysed by the chloroplast Hydrogenase (HydA). This separation was achieved by culturing C. reinhardtii first in the presence of sulfur to build stores of an endogenous substrate and then in the absence of sulfur. Sulfur is required for the de novo synthesis of the D1 protein of the PSII reaction centre, and of course of many other organic components of the cell. The D1 protein has an approximate half-life of 30 min, being damaged under non-optimal conditions through the highly oxidizing reaction that it drives. In the presence of sulfur, high levels of active PSII are maintained and H2O is split into H+, e− and O2. As sulfur levels decrease in a sulfur depleted medium the H+ and e− are subsequently recombined by HydA to generate H2, which lifts the inhibition of the Hydrogenase induced by O2. This, for the first time, facilitated long-term H2 production using wild type (WT) C. reinhartii. 
The Melis process is, however, subject to considerable practical constraints. The actual rate of hydrogen gas accumulation is at best 15 to 20% of the photosynthetic capacity of the cells (Melis and Happe 2001) and suffers the inherent limitation that hydrogen production by S deprivation of the algae cannot be continued indefinitely. The yield begins to level off and decline after about 40-70 hours of S deprivation, and after about 100 hours of S deprivation the algae need to revert to a phase of normal photosynthesis to replenish endogenous substrates.
International Publication No. WO 03/067213 describes a process for hydrogen production using Chlamydomonas reinhardtii wherein the algae has been genetically modified to down regulate expression of a sulfate permease, CrcpSulP, through insertion of an antisense sequence. This is said to render obsolete prior art sulfur deprivation techniques, as it obviates the need to physically remove sulfur nutrients from growth media in order to induce hydrogen production. The reduced sulfur uptake by the cell using this technique not only results in a substantial lowering of the levels of the major chloroplast proteins such as Rubisco, D1 and the LHCII, but also deprives the cell of sulfur for use in the biosynthesis of other proteins.
Consequently there remains a need to identify a sustainable and efficient process for photosynthetic hydrogen production that avoids sulfur deprivation.